Method of Quantifying Cholesterol in High Density Lipoprotein and Reagent Compositions

ABSTRACT

A method for specifically quantifying HDL cholesterol in which cholesterol in lipoproteins other than HDL is erased in the first step, and HDL cholesterol is specifically quantified in the second step, by which accurate values can be obtained even in measurements of abnormal samples such as disorder of lipid metabolism and lipoprotein abnormality, is disclosed. The method for quantifying cholesterol in high density lipoprotein according to the present invention comprises a first step of erasing cholesterol in lipoproteins other than high density lipoprotein by treating a test sample with cholesterol esterase and cholesterol oxidase in the absence of a surfactant which acts on high density lipoprotein and removing generated hydrogen peroxide; and a second step of adding a surfactant which specifically acts on high density lipoprotein to the product of said first step and quantifying hydrogen peroxide generated from cholesterol in high density lipoprotein by actions of cholesterol esterase and cholesterol oxidase. As the cholesterol oxidase used in the first step, one having a molecular weight of not more than 60 kilodaltons is used.

This application is a Divisional of co-pending application Ser. No.10/502,959 filed on Sep. 29, 2004 and for which priority is claimedunder 35 U.S.C. § 120. Application Ser. No. 10/502,959 is the nationalphase of PCT International Application No. PCT/JP03/00867 filed on Jan.30, 2003 under 35 U.S.C. § 371. The entire contents of each of theabove-identified applications are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for quantifying cholesterol inhigh density lipoprotein (HDL) and to a reagent composition usedtherefor.

BACKGROUND ART

It is known that HDL relates to removal of cholesterol accumulated incells because it receives cholesterol from various tissues includingwalls of blood vessels with arterial sclerosis, so that HDL is usefulfor estimating the risk for various arterial sclerosises includingcoronary artery sclerosis, and that its blood level is an indicator forthe risk of onset of arterial sclerosis.

Methods for measuring cholesterol in HDL include a method in which HDLis separated from other lipoproteins by ultracentrifugation and then theHDL is measured; and a method in which the cholesterol in HDL isseparated by electrophoresis, then the lipid is stained, and theintensity of the generated color is measured. However, these methods arecomplex or a number of samples cannot be assayed, so that they are notcommonly used.

The method for measuring the cholesterol in HDL, which is generally usedin the field of clinical test is the method in which a precipitatingagent is added to the sample so as to coagulate the lipoproteins otherthan HDL, removing the coagulated lipoproteins by centrifugation, andthe cholesterol in the resulting supernatant containing HDL alone ismeasured. Although this method is simpler than the ultracentrifugationmethod and the electrophoresis method, it is not satisfactorily simplebecause it comprises addition of the precipitating agent and subsequentseparation, and a comparative large amount of sample is needed.

On the other hand, methods in which the cholesterol in HDL is separatelyquantified by using enzymes have been proposed. For example, a method isknown, which comprises the steps of preliminarily coagulating thelipoproteins other than HDL by an antibody and polyanion, enzymaticallyreacting the cholesterol in HDL alone, inactivating the enzyme andsimultaneously re-dissolving the coagulated mass, and measuring theabsorbance of the resulting solution (Japanese Laid-open PatentApplication (Kokai) No. 6-242110). However, this method has a problem inthat it is necessary to add reagents at least three times, so that thismethod can be practiced only by the limited analyzing apparatuses.Therefore, this method is not widely used.

Other methods include a method in which an enzyme reaction is carriedout in the presence of a bile salt or a nonionic surfactant (JapaneseLaid-open Patent Application (Kokai) No. 63-126498); a more recentlydeveloped method in which the cholesterol in HDL is specifically trappedby chemically modified cholesterol esterase and/or cholesterol oxidasein the presence of a clathrate compound such as cyclodextrin (JapaneseLaid-open Patent Application (Kokai) No. 7-301636); and a method inwhich the lipoproteins other than HDL are made into aggregates orcomplexes and then the cholesterol in HDL is trapped by an enzymereaction (Japanese Laid-open Patent Application (Kokai) Nos. 8-131197and 8-201393). However, with these methods, the results for certainsamples are different from the results by the precipitation method, sothat their specificities are problematic.

The present applicant previously developed a method for quantifying HDLcholesterol which does not necessitate a fractionating operation(International Publication No. WO98/26090), and the reagent therefor isnow being generally used in the actual clinical tests. In this method,cholesterol in lipoproteins other than HDL in a sample is erased (theterm “erase” herein means to decompose ester type cholesterol and freecholesterol, and to make the decomposed products undetectable in thesecond step), and HDL cholesterol is specifically quantified in thesecond step.

However, this method has a problem in that the measured amount of HDL islarger than the actual amount of HDL for abnormal clinical samples suchas disorder of lipid metabolism and lipoprotein abnormality. Abnormalsamples often indicate abnormal triglyceride (TG) values, bilirubinvalues and the like in biochemical tests, so that overcoming theabove-mentioned problem will increase the usefulness of the measuringmethod, and so the solution of the problem is demanded.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide a methodfor quantifying HDL cholesterol in which cholesterol in lipoproteinsother than HDL is erased in the first step, and HDL cholesterol isspecifically quantified in the second step, by which accurate values canbe obtained even in measurements of abnormal samples such as disorder oflipid metabolism and lipoprotein abnormality.

The present inventors investigated the cause of the positive error ofmeasurement to discover that, with the abnormal samples, the cholesterolin the lipoproteins other than HDL is not well erased in the first step,and the cholesterol is carried over to the HDL-specific reaction in thesubsequent second step, thereby giving a positive influence to thereaction of HDL.

Thus, the present inventors studied the method for increasing the degreeof erasing in the first step to discover that the degree of erasing ofcholesterol in the lipoproteins other than HDL is increased by using acholesterol oxidase having a small molecular weight in the first step.

More particularly, lipoprotein particles are formed by aggregation ofester type cholesterol, free cholesterol, TG (triglyceride),phospholipids and proteins. Each particle has a structure in whichproteins and phospholipids exist at the surface of the particle, freecholesterol exists therein, and ester type cholesterol and TG exist atthe center. By applying a cholesterol oxidase having a small molecularweight to the lipoproteins, the cholesterol oxidase can enter the insideof the lipoproteins other than HDL and reacts with free cholesterolexisting near the surface to change the particle structure, so thatcholesterol esterase also acts on the ester type cholesterol, therebypromoting the erasing reaction. In this case, since HDL is a highdensity lipoprotein and the percentage of proteins at the surface ishigh, the low molecular cholesterol oxidase cannot enter the inside ofthe particle, so that the reaction does not occur.

That is, the present invention provides a method for quantifyingcholesterol in high density lipoprotein, comprising a first step oferasing cholesterol in lipoproteins other than high density lipoproteinby treating a test sample with cholesterol esterase and cholesteroloxidase in the absence of a surfactant which acts on high densitylipoprotein and removing generated hydrogen peroxide; and a second stepof adding a surfactant which specifically acts on high densitylipoprotein to the product of the first step and quantifying hydrogenperoxide generated from cholesterol in high density lipoprotein byactions of cholesterol esterase and cholesterol oxidase; characterizedin that the cholesterol oxidase used in the first step has a molecularweight of not more than 60 kilodaltons. The present invention alsoprovides a reagent composition for quantifying cholesterol in highdensity lipoprotein, which is used for the first step of theabove-described method according to the present invention, whichcomprises the cholesterol esterase, the cholesterol oxidase having amolecular weight of not more than 60 kilodaltons and a component whichremoves hydrogen peroxide.

By the method of the present invention, in a method for measuring HDL ina test sample containing HDL and other lipoproteins such as LDL, VLDLand CM, HDL may be selectively, simply and accurately quantified evenwhen the sample is an abnormal test sample such as high TG or one from apatient suffering from a hepatic disorder (high bilirubin).

BEST MODE FOR CARRYING OUT THE INVENTION

Cholesterols contained in lipoproteins include ester type cholesterol(cholesterol ester) and free cholesterol. In this-specification, theterm “cholesterol” includes both of these unless otherwise specified.

The test sample subjected to the method of the present invention may beany sample which may contain lipoproteins such as HDL, LDL, VLDL and CM.Examples of the test samples include body fluids such as blood, sera andplasma as well as dilutions thereof although the test samples are notrestricted thereto. The method of the present invention is particularlyuseful when the test sample is an abnormal sample such as a blood samplecontaining TG at a level of not less than 400 mg/dL, particularly notless than 1000 mg/dL, or containing bilirubin at a level of not lessthan 2.00 mg/dL, particularly not less than 3.00 mg/dL. These values areblood levels of non-diluted blood. As mentioned above, by the knownmethod, there is a problem in that the determined amount of HDL, whichis often measured for samples containing high TG or high bilirubin, ishigher than the actual amount of the HDL. As will be concretely shown inExamples below, by the method of the present invention, the amount ofHDL can be accurately measured even when the sample is a blood samplecontaining high TG or high bilirubin. It should be noted that “bloodsample” herein includes whole blood, serum and plasma, as well asdilutions thereof.

The method of the present invention comprises a first step and a secondstep. In the first step, the cholesterol in LDL, VLDL and CM is erasedin the absence of a surfactant which acts on HDL. In the subsequentsecond step, cholesterol is quantified using a surfactant specific toHDL. As the cholesterol oxidase used in the first step, one having a lowmolecular weight is used.

The term “erase” in the first step means to decompose cholesterol, andto make the decomposed products undetectable in the second step. Themethods for selectively erasing the cholesterol in the lipoproteinsother than HDL, that is, in LDL, VLDL, CM and the like include thefollowing methods.

That is, cholesterol esterase and cholesterol oxidase are acted on thetest sample in the absence of a surfactant which acts on HDL, and thegenerated hydrogen peroxide is removed. By the action of cholesterolesterase, the ester type cholesterol in the lipoproteins are hydrolyzedto yield free cholesterol and fatty acids. The thus generated freecholesterol and the free cholesterol inherently existing ill thelipoproteins are oxidized by the action of cholesterol oxidase to yieldcholestenone and hydrogen peroxide. The thus generated hydrogen peroxideis removed. Methods for removing hydrogen peroxide include a method inwhich the hydrogen peroxide is decomposed to water and oxygen bycatalase; and a method in which a phenol-based or aniline-based hydrogendonor compound, such as DAOS(N-ethyl-N-(2-hydroxysulfopropyl)-3,5-dimethoxyaniline), which reactswith hydrogen peroxide to yield a colorless quinone, is reacted with thehydrogen peroxide to convert the hydrogen peroxide to the colorlessquinone, although the methods for removing hydrogen peroxide are notrestricted to these methods.

In the above-mentioned first step, by treating the sample with the lowmolecular cholesterol oxidase in the absence of a surfactant which actson HDL, the cholesterol in HDL is not substantially reacted, while thecholesterol in the other lipoproteins such as LDL, VLDL and CM arereacted and erased. By this, in the subsequent second step, thecholesterol in HDL is selectively quantified.

The molecular weight of the cholesterol oxidase used in the first stepis 20 kDa to 60 kDa, preferably 30 kDa to 40 kDa. The cholesteroloxidase having a molecular weight within this range, which is used inthe method of the present invention, may be obtained from variousmicroorganisms such as bacteria and yeasts, and its origin is notrestricted at all. Further, since such a cholesterol is commerciallyavailable, a commercially available one may be employed. In the knownmethods for measuring HDL, high molecular cholesterol oxidases havingmolecular weights of more than 60 kilodaltons have been employed.

The concentration of the cholesterol esterase in the reaction mixture inthe first step may preferably be about 0.2 to 2.0 U/mL, and cholesterolesterases produced by bacteria belonging to genus Pseudomonas areeffective. The concentration of the cholesterol oxidase may preferablybe about 0.1 to 1.5 U/mL. In cases where catalase is used as a componentfor removing hydrogen peroxide, the concentration of the catalase maypreferably be about 50 to 2000 U/mL. The concentration of the peroxidaseused for converting hydrogen peroxide to colorless quinone maypreferably be about 0.4 to 1.0 U/mL. The concentration of thephenol-based or aniline-based hydrogen donor compound may preferably beabout 0.4 to 2.0 mmol/L.

The reaction in the first step is carried out in a buffer with a pH of 5to 8. The buffer may preferably be phosphate buffer, glycine buffer,Tris buffer or Good's buffer. Especially, Bis-Tris, PIPES, MOPSO, BES,HEPES and POPSO which are Good's buffer are preferred. The concentrationof the buffer may preferably be about 10 to 500 mM.

A lipoprotein hydrolase may optionally be added to the reaction mixturein the first step. Addition of this enzyme is preferred becauseespecially the cholesterol in VLDL easily reacts. The concentration ofthis enzyme in the reaction mixture may preferably be about 5.0 to 10.0U/mL. Further, the reaction solution in the first step may optionallycontain a surfactant which does not substantially act oil HDL aid/orother component(s) such as clathrate compounds including cyclodextrin,in (an) amount(s) not adversely affecting the effect of the presentinvention.

The reaction temperature in the first step may preferably be about 25°C. to 40° C., and 37° C. is most preferred. The reaction time may beabout 2 to 10 minutes.

In the following second step, a surfactant which specifically acts onHDL is added to the reaction product of the first step, and thecholesterol in high density lipoprotein is enzymatically quantified. Theterm “surfactant which specifically acts on HDL” means a surfactant bywhich the cholesterol in HDL reacts due to the action of enzymes such ascholesterol esterase and cholesterol oxidase (the reaction ratio is notless than 70%, preferably not less than 90%), while the cholesterol inthe lipoproteins other than HDL does not substantially react (thereaction ratio is not more than 30%, preferably not more than 20%). Thehydrophilicity lipophilicity balance (HLB) of the surfactant used hereis 13 to 14. As the surfactant, nonionic surfactants are preferred, andpolyalkylene oxide derivatives are especially preferred. Among thepolyalkylene oxide derivatives, polyethylene oxide derivatives are mostpreferred. The above-mentioned range of HLB may be attained by mixing aplurality of surfactants, and such a mixture of a plurality ofsurfactants may also be used. The method for calculating HLB ofsurfactants is well-known, and is described in, for example, HiroshiHORIGUCHI, “New Surfactants”, 1986, Sankyo Shuppan.

Preferred specific examples of the surfactant include polyoxyethylenelauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether,polyoxyethylene higher alcohol (C4-C35) ether, polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxybenzylphenylether and the like, although the surfactant is not restricted thereto.

Although the concentration of the surfactant in the second step is notrestricted, it may preferably be 0.05 to 3% by weight, more preferably0.1 to 1.5% by weight based on the total reaction mixture.

In the presence of the above-mentioned surfactant, the HDL cholesterolin the test sample may be enzymatically quantified. That is, in thefirst step, most of the cholesterol in the lipoproteins other than HDLis erased, and with the synergistic effect with the reaction in thesecond step, the cholesterol in HDL alone is quantified.

The method for enzymatically quantifying cholesterol per se iswell-known in the art. For example, as in the first step, cholesterol isquantified by generating hydrogen peroxide from cholesterol ester andfree cholesterol by the actions of cholesterol esterase and cholesteroloxidase, and by quantifying the generated hydrogen peroxide.Quantification of hydrogen peroxide may be carried out by, for example,reacting the hydrogen peroxide with a compound which forms a quinonepigment, and by measuring the amount of the generated quinone pigment bymeasuring absorbance or the like. The quinone pigment may be formed by,for example, reacting hydrogen peroxide and 4-aminoantipyrine and DAOSor HDAOS (N-(2-hydroxysulfopropyl)-3,5-dimethyoxyaniline). The quinonepigment formed thereby has the maximum absorbance at 593 nm when DAOS isused, and has the maximum absorbance at 583 nm when HDAOS is used.Although the concentration of the compound which yields the quinonepigment is not restricted, the concentration of 4-aminoantipyrine, forexample, may preferably be 0.1 to 2.0 mM, more preferably 0.5 to 1.5 mM,and the concentration of DAOS or HDAOS may preferably be 0.1 to 1.5 mM,more preferably 0.4 to 1.0 mM. Although the concentration of theperoxidase is not restricted, it may preferably be 0.4 to 5 U/mL in thetotal reaction mixture. Preferred reaction conditions (reactiontemperature, reaction time, buffer and pH) are the same as the preferredreaction conditions in the first step.

In cases where the generated hydrogen peroxide is decomposed withcatalase, a catalase inhibitor such as sodium azide is used in thesecond step so as to inhibit the catalase because it is necessary toinhibit the catalase in the second step.

The present invention also provides a reagent composition forquantifying cholesterol in high density lipoprotein, which is used forthe first step of the method of the present invention, which comprisescholesterol esterase, cholesterol oxidase having a molecular weight ofnot more than 60 kilodaltons and a component which removes hydrogenperoxide. As the component which removes hydrogen peroxide, as mentionedabove, (1) catalase or (2) phenol-based or aniline-based hydrogen donorcompound and peroxidase, or the like may be employed. The ratio of thecomponents in the reagent composition is the ratio with which theabove-mentioned concentrations are attained when used. The reagentcomposition may further comprise the above-mentioned buffer agent and/orthe lipoprotein hydrolase.

The present invention will now be described more concretely by way ofexamples thereof. It should be noted, however, the present invention isnot restricted to the examples below. In the examples below, all “%” areby weight unless otherwise specified.

REFERENCE EXAMPLE

Using samples containing known amounts of purified HDL, LDL, VLDL andCM, respectively, the cholesterol in each of the lipoproteins wasenzymatically quantified in the presence of a nonionic surfactantEmulgen 911 (polyoxyethylene nonyl ether, HLB 13.7), Emulgen B66(polyoxyethylene derivative, ALP 13.2) or a mixture of Emulgen B66 andEmulgen A90 (polyoxyethylene derivative, HLB 14.5), all of which arecommercially available from KAO CORPORATION. This operation was carriedout as follows.

To a solution containing 0.5 U/mL of cholesterol esterase, 0.4 U/mL ofcholesterol oxidase, 0.5 U/mL of peroxidase, 1.0 n-mol/L of4-aminoantipyrine and 0.5 mmol/L of HDAOS in 50 mM PIPES buffer, pH 7.0,Emulgen 911 or Emulgen B66 was added to a concentration of 0.1% byweight, or Emulgen B366/Emulgen A90 mixture (9/1) was added to aconcentration of 1.3% by weight. Twenty microliters of each sample wasmixed with 2.0 mL of the thus prepared reagent and the resulting mixturewas allowed to react at 37° C. for 10 minutes, followed by measuringabsorbance at 600 nm.

As a result, the reaction ratio (i.e., the ratio of the quantifiedcholesterol in the total cholesterol) was about 95% for the cholesterolin HDL, and about 18 to 22% for the cholesterols in other lipoproteins.

From this, it can be seen that Emulgen 911, Emulgen B66 and the EmulgenB66/Emulgen A90 mixture are within the scope of the term “surfactantwhich specifically acts on high density lipoprotein”.

Example 1

Three types of reagents having the following compositions were prepared:

In the three types of reagents, only the molecular weight of cholesteroloxidase was different, and all of the other components were the same.

<Common Components> First Reagent BES buffer, pH 7.0 100 mmol/L HDAOS0.7 mmol/L Cholesterol esterase 1.5 U/mL Catalase 80 U/mL Second ReagentBES buffer, pH 7.0 100 mmol/L 4-aminoantipyrine 4.0 mmol/L Peroxidase4.0 U/mL Sodium azide 0.1% Emulgen B66 (HLB13.2) commercially 1.3%available from KAO CORPORATION

<Reagent>

One of cholesterol oxidases (CO) having different molecular weights wasadded to the first reagent of each reagent to a concentration of 0.8U/mL.

Reagent 1

High molecular CO (trademark: CON II, commercially available from AsahiKasei Corporation, molecular weight: 61.8 kDa)

Reagent 2

Low molecular CO (trademark: COO-321, commercially available from ToyoboCo., Ltd., molecular weight: 55.0 kDa)

Reagent 3

Low molecular CO (trademark: CO, commercially available from Asahi KaseiCorporation, molecular weight: 38.0 kDa)

Reagent 4

Low molecular CO (trademark: COO-311, commercially available from ToyoboCo., Ltd., molecular weight: 34.0 kDa)

To 4 μL of each of the samples (sera) from healthy individuals, samplesof high TO and samples of high bilirubin (BIL), 300 μL of theabove-described first reagent preliminarily warmed at 37° C. was addedand the mixture was allowed to react at 37° C. for 5 minutes. The 100 μLof the second reagent was added and the mixture was allowed to react at37° C. for 5 minutes, followed by measurement of absorbance at 600 nm ofthe reaction solution. On the other hand, the amounts of HDL cholesterolin the same samples were determined by the ultracentrifugation methoddescribed in “New Biochemistry Experiments Lecture”, Vol. 4, Lipid I,Triglycerides and Lipoproteins”, 181 (1993). The percentage of thedifference between the value determined by using each reagent and thevalue determined by the ultracentrifugation method was calculated.

(A−B)/B×100

(wherein A represents the value obtained by using each reagent, and Brepresents the value obtained by the ultracentrifugation method).

The concrete methods for measuring the TG value and the BIL value wereas follows:

As the method for measuring TO value, the LPL-GK-GPO-based enzyme methoddescribed in “Clinical Test Handbook, vol. 31”, 559 (1998) was employed.The BIL value was measured by the Michaelsson's modified method which isa modification of Jendrassik-Grof method described in “Clinical TestHandbook, vol. 31”, 559 (1998).

The results are shown in Tables 1 to 3. The values in the parenthesesindicate the percentage of the difference from the value obtained by theultracentrifugation method, which percentage was calculated by theabove-described equation.

TABLE 1 Samples from healthy individuals Ultracentrifugation MethodReagent 1 Reagent 2 Reagent 3 Reagent 4 1 45.0 45.3(0.7) 44.2(−1.8)45.3(0.7) 44.9(−0.2) 2 53.4 54.0(1.1) 54.5(2.1) 53.7(0.6) 54.1(1.3) 366.7 66.6(−0.1) 66.5(−0.3) 66.4(−0.4) 66.8(0.1) 4 78.3 77.8(−0.6)79.3(1.3) 78.5(0.3) 78.6(0.4) 5 85.7 86.4(0.8) 85.3(−0.5) 85.9(0.2)85.9(0.2) unit: mg/dL (difference: %)

TABLE 2 Samples of high TG values Ultracen- trifugation Method Reagent 1Reagent 2 Reagent 3 Reagent 4 1 (TG value 43.0 47.8(11.2) 44.9(4.4)42.9(−0.2) 43.0(0.0) 1230) 2 (TG value 30.3 35.0(15.5) 31.7(4.6)29.7(−2.0) 29.8(−1.7) 1560) 3 (TG value 36.0 38.7(7.5) 36.7(1.9)35.9(−0.3) 36.0(0.0) 1870) 4 (TG value 24.6 26.9(9.3) 25.8(4.9)25.0(1.6) 24.8(0.8) 2040) 5 (TG value 33.2 35.2(6.0) 34.1(2.7) 33.4(0.6)33.2(0.0) 2600) unit: mg/dL (difference: %)

TABLE 3 Samples of high BIL values Ultracen- trifugation Method Reagent1 Reagent 2 Reagent 3 Reagent 4 1 (BIL value 60.1 70.1(16.6) 65.8(9.5)61.9(3.0) 60.5(0.7) 3.3) 2 (BIL value 14.2 22.4(57.7) 15.7(10.6)13.9(−2.1) 13.8(−2.8) 5.6) 3 (BIL value 13.1 36.4(178) 19.4(48.1)12.2(−6.9) 12.3(−6.1) 8.2) 4 (BIL value 10.6 20.1(89.6) 11.8(11.3) 9.8(−7.5)  9.6(−9.4) 12.4) 5 (BIL value 5.2 16.2(212)  9.9(90.4) 5.2(0.0)  5.1(−1.9) 16.8) unit: mg/dL (difference: %)

As shown in Table 1, for the samples from healthy individuals, valuessimilar to the values determined by the ultracentrifugation method wereobtained when any of the reagents was used, while for the high TG orhigh BIL samples, the values obtained by using a low molecularcholesterol oxidase was closer to the values obtained by theultracentrifugation method.

1. A reagent composition for quantifying cholesterol in high densitylipoprotein, said composition comprising cholesterol esterase andcholesterol oxidase having a molecular weight of not more than 55.0kilodaltons and a component which removes hydrogen peroxide.
 2. Thecomposition according to claim 1, wherein said cholesterol oxidase has amolecular weight of 30 to 40 kilodaltons.
 3. The composition accordingto claim 1 or 2, wherein said component which removes hydrogen peroxideis catalase.
 4. The composition according to claim 1 or 2, wherein saidcomponent which removes hydrogen peroxide is a phenol-based oraniline-based hydrogen donor compound which reacts with hydrogenperoxide to yield a colorless quinone.
 5. The composition according toclaim 4, wherein said phenol-based or aniline-based hydrogen donorcompound is N-ethyl-N-(2-hydroxysulfopropyl)-3,5-dimethoxyaniline.